NSC 650426D (KRN5500) is a semisynthetic spicamycin derivative which showed potent, selective growth inhibitory activity against many of the colon, ovarian and renal cell lines in the NCI primary in vitro anticancer drug screen. In addition, in vivo antitumor activity has been demonstrated in a variety of xenograft models. The objectives of this project were to develop an analytical method for the assay of NSC 650426D in biological fluids and to define the in vivo pharmacokinetic properties of the compound in order to provide an objective basis for selection and optimization of the route of administration, dose and dose schedule. An analytical method was developed for the assay of NSC 650426D in plasma which employed reversed phase HPLC with UV detection. Using 50 micro l of plasma, concentrations of 0.1 micrometer (0.06 mg/ml) were assayed with acceptable reproducibility. Plasma concentration-time profiles following IV injection of mice with 42.5 and 21.2 micro m/kg (50 and 25 mg/kg), and of dogs with 11.9 and 5.9 micro m/kg (7 and 3.5 mg/kg) were fitted by computerized nonlinear regression analysis. Profiles exhibited biexponential behavior in the mouse, and triexponential behavior in the dog. While mean initial half- lives were rapid in both the mouse and dog (approximately 14 and 5 minutes, respectively), the mean terminal half-life in the mouse was much more rapid than that in the dog (102 minutes vs. 14 hours). The mean plasma total body clearance in the mouse was also significantly more rapid than in the dog (12 vs. 2 ml/min/kg). The large total body volumes of distribution in both the mouse and dog (1.8 and 2.5 l/kg) suggest that the compound is highly distributed into deep tissue sites. Following IP administration in mice with a dose of 51 micro m/kg (30 mg/kg), plasma levels increased to approximately 2 micro M at 2 hours following dosing, then declined to about 0.2 micro M after 4 hours, and remained in the range from 0.2 to 0.4 micro M through 24 hours. An estimate of the bioavailability of the compound from the peritoneal cavity during 24 hours after dosing was 16%. These plasma concentrations are in excess of those which resulted in growth inhibition in vitro.